Highly Multiplexed Nanoscale Imaging Platforms for Profiling and Interrogation of Complex Diseases. Yongxin Zhao Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA. Abstract Our bodies are configurations of molecular building blocks (e.g., proteins, nucleic acids, lipids, and carbohydrates) that are assembled with nanoscale precision. Misconfiguration of these building blocks is a hallmark of many diseases, such as cancer, infection, and inflammation. Characterization of such misconfigurations in nanoscale is critical for understanding pathogenesis of complex diseases as well as their accurate diagnosis and efficient development of therapeutics. In modern pathology, diffraction-limited microscopy is used to examine biopsies. However, diffraction-limited microscopy is unable to resolve a large number of distinct molecular species and their nanoscale configurations, which severely limited its capability of analyzing intricate and subtle pathological changes. To enable pathology-optimized nanoscopy, I recently developed a technique, termed Expansion Pathology (ExPath), that supports economical, nanoscale morphological imaging of biopsies by magnifying them physically, rather than optically, and that requires only diffraction-limited microscopes. ExPath opens many new venues in the field of pathology. To enable highly multiplexed nanoscale in situ analysis of molecular misconfigurations associated with complex diseases, I propose three independent ExPath-derived imaging platforms, built upon new chemical and imaging strategies: (1) Tissue proteome-preserving swellable polymer framework for serial, multiplexed nanoscale imaging; (2) Highly multiplexed, error-robust barcoded imaging system for RNA and proteins; and (3) Expansion microbiology imaging platform for bacterial/fungal infectious diseases. We will collaborate with experts and end users, such as pathologists, clinicians and microbiologists, to pressure test these technologies on diverse applications from precancerous breast lesions to brain tumors to infected tissues. This new portfolio of ExPath- based techniques will enable profiling and analysis of cell-type heterogeneity and intricate molecular interactions with nanoscale precision, and will extend the range of applicable sample types, covering pathogens, biofilms and infected tissues. These studies will generate a diversity of tools with broad applicability in pathology, and throughout biology. We will continue our commitment to distribute the tools we develop freely and to instruct the community on their usage. 1